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SNPsaurus 05-12-2015 04:06 PM

Brian, when we were developing local assembly of paired-end RAD, we were surprised to see contigs of 1200 bp being assembled (see http://journals.plos.org/plosone/art...l.pone.0018561 figure 4), meaning that there must have been fragments of 1200 bp undergoing bridge amplification. We had to use a "triangle cut" in the gel size selection to over-represent the larger fragments, but they did bridge.

I think the size preference in the patterned flow cells could be because a small fragment could enter a well after a larger fragment but then outcompete the larger fragment to fill the well. Or in the diffusion kinetics?

pmiguel 05-13-2015 06:33 AM

Quote:

Originally Posted by Brian Bushnell (Post 172315)
Impressive; I was under the impression that inserts much over 800bp simply would not bridge-amplify. Maybe we should try that approach! Anyway, rather than shorter molecules vastly out-competing longer molecules at all lengths, that could be a more of a case where the rates are fairly similar up to a point (1kbp?) after which longer molecules start failing to form clusters at all (even if there were no short molecules present). I'm just guessing, though.

We did cluster at 1/2 the normal density, so that may have allowed the longer amplicons to form clusters where normally they would not have. Again, my natural inclination is to regard this as some sort of competition. Looking at plots of insert sizes and comparing them to the sizes of the input library it has always looked to me as if all the amplicons queued up by length and then all the shortest ones clustered. Okay, an exaggeration, but more-or-less fitting what one sees.

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Phillip

DNATECH 05-21-2015 12:24 AM

The latest HiSeq3000 run (we did receive a few flowcells) did average 378 million clusters passing filter, per lane. All libraries were size selected.

One obvious part of the exclusion amplification as implemented is the very viscous enzyme mix. Probably the diffusion of the library fragments towards the flowcell is very much slowed down (requiring also higher library concentrations?) giving the molecule that arrives first the chance to become amplified and fill entire nanowells before a second one arrives (http://www.google.com/patents/WO2013188582A1?cl=en). The viscosity enhanced "drag" also could explain the stronger bias towards smaller inset size reads?
The high viscosity buffer together with high library concentrations and "RPA" amplification for the clustering process ("Recombinase Polymerase Amplification" ( http://www.twistdx.co.uk/our_technology/ )) might be sufficient for the Kinetic Exclusion Amplification on the nanowell flowcells? It seems to me that the other methods described in the patent might not be compatible with the old cBots (these can be used for the Hiseq3000/4000 clustering after a software upgrade)?


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